Summary: The overall objective of this proposal is to expand from co-culture to tri-culture of the pathogenic yeast, Candida albicans, and bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, with unprecedented spatiotemporal resolution and the ability to meet different culture requirements in an effort to model the complexity observed in microbiome. In nature, microbial life occurs in a concourse where interactions such as cell-cell signaling or metabolite trading between the same species and often across kingdoms are key to their survival. In human respiratory and gastrointestinal tracts, the balance between competition and cooperation between fungi and bacteria is of particular importance as these interactions can determine the outcome of highly invasive infections. Microbial communities often grow in matrices called biofilms with intricate spatial structure, and many coexist as micro-colonies separated by a few hundred micrometers. This spatial structure has been hypothesized to be important in microbial ecology. Modeling the microbial interactions in a well-controlled and spatially analogous manner is of great interest in microbiome engineering and developing new biological technologies. The vast majority of microbial models or the microbe-host systems, however, are limited to mixed or binary cultures that either are challenging to track changes occurring in individual populations, or lack the compatibility to support different nutrient and environmental requirements for different species. In particular, co-culturing anaerobic and aerobic bacteria is impossible with current technologies. Here, the PI proposes a unique ?fluitrode? platform that circumvents the aforementioned bottlenecks. The research will test four hypotheses: (1) Different microbe species cultured in a spatially controlled manner have significant advantages over standard mixed cultures; (2) Effect of spatial resolution on the communication between C. albicans and P. aeruginosa can be revealed using the fluitrode platform; (3) Differences in microbial communication between aerobic and anaerobic states can be revealed with the heterogeneous culturing platform; and (4) The expansion to tri-culture C. albicans, P. aeruginosa and S. aureus can lead to the construction of a synthetic microbiome of many more species with individual culturing media, optimal spatial resolution, and heterogeneous oxygen requirements. Successful culturing of the three species will lead to establishing synthetic microbiome with more complexity as well as more controllability that is impossible with other approaches. In the short term, the synthetic microbiome will expedite scientific communities to understand the intricate cell-cell interactions in native microbiome. In the long term, better models of polymicrobial interactions will pave the way to developing better treatments for microbial-based diseases, which is a major public health concern.